Tetraploid watermelon line 34wa009

ABSTRACT

The present invention provides novel watermelon line 34WA009 and plant parts, seed, and tissue culture therefrom. The invention also provides methods for producing a watermelon plant by crossing the watermelon plants of the invention with themselves or another watermelon plant. The invention also provides watermelon plants produced from such a crossing as well as plant parts, seed, and tissue culture therefrom. Further provided are methods of producing triploid watermelon seed and plants and seedless watermelon fruit produced therefrom as well as the triploid watermelon seed and plants and the seedless fruits produced by such methods.

FIELD OF THE INVENTION

This invention is in the field of watermelon plants, in particular, theinvention relates to a novel tetraploid watermelon line, which can beused to produce triploid seeds and plants for production of seedlesswatermelon fruit.

BACKGROUND OF THE INVENTION

This invention relates to a new and unique inbred tetraploid watermelonline, designated 34WA009.

Watermelon is an important horticultural crop that accounts for 2% ofthe world area devoted to vegetable crops. There were 3,810,535 hectares(Ha) of watermelon grown in the world, and 51,110 Ha of watermelonsgrown in the United States in 2009. Asia is by far the most importantwatermelon production site with 78% of the world area and 83.4% of theworld production of 100,687,056 metric tons. The estimated annual worldwatermelon value exceeded $7.6 billion when using the United Statesaverage price for 1995-1997. Watermelon is grown in at least forty-fourstates in the United States, with Florida, Georgia, California, andTexas, having long warm growing seasons, being the major producingstates. In the United States, watermelon production has increased from1.2 M tons in 1980 to 3.8 M tons in 2009, with an annual farm value of$470 million (U. S. Department of Agriculture, Agricultural Statistics,2009).

In recent years, there has been an increase in consumer demand forseedless watermelons, and production of seedless watermelon hasincreased significantly. Triploid seedless watermelons have beencommercially grown in the United States since the late 1980's.Currently, over 80% of the watermelons produced in the United States aretriploid seedless watermelons. Seedless watermelon receives well abovethe average price for seeded watermelons in the market. Triploidseedless watermelon also produces higher yields than the diploid seededwatermelons.

Triploid seedless watermelon is a true F1 hybrid between a tetraploidwatermelon, as the female parent, and a diploid watermelon, as the maleparent (Kihara, H. 1951, Triploid Watermelons, Proceedings of AmericanSociety for Horticultural Science, 58:217-230). Diploid watermelons have22 chromosomes (2N=2X=22) in their somatic cells, and tetraploidwatermelons have 44 chromosomes (2N=4X=44) in their somatic cells. Cellswith three sets of homologous chromosomes are said to be triploid andare designated as 3X. When female flowers of tetraploid plants are crosspollinated by the male flowers of diploid plants, the fruits produced bythe tetraploid plants contain triploid seeds that produce triploidplants. The triploid seedless watermelons have 33 chromosomes (2N=3X=33)in their somatic cells. When the triploid plants are grown with diploidplants in the same field, the triploid plants produce fruits that areseedless. The seedless condition in triploid watermelon is the result ofthe presence of three homologous sets of chromosome per somatic cellrather than the usual two. The inability of the triploid zygote toproduce normal viable gametes (pollen and egg cells) causes the absenceof seeds in triploid fruits. Typically, seedless watermelons containsmall edible white ovules, similar to those in immature cucumbers.

Watermelon, in general, and seedless watermelon in particular, is animportant and valuable vegetable crop. Thus, there is an ongoing needfor improved inbred tetraploid watermelon lines.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred tetraploidwatermelon line designated 34WA009, characterized by producing maturefruits having a round shape, a Crimson Sweet striping pattern, mediumseed size, large fruit size, firm flesh texture, an intense red fleshcolor, an intermediate rind thickness, and having resistance to FusariumWilt race 1, Powdery Mildew race 1, and Anthracnose race 1. As comparedwith tetraploid line 4XCS34, the mature fruits of line 34WA009 have ahigher plant vigor as well as a greater maximum fruit weight. Theinvention also encompasses the seeds of watermelon line 34WA009, theplants of watermelon line 34WA009, plant parts of the watermelon line34WA009 (including fruit, seed, gametes, scion, rootstock, shoots),methods of producing seed from watermelon line 34WA009, and methods forproducing a watermelon plant by crossing the watermelon line 34WA009with itself or another watermelon plant, methods for producing awatermelon plant containing in its genetic material one or moretransgenes, and the transgenic watermelon plants produced by thatmethod. The invention also relates to methods for producing otherwatermelon plants derived from watermelon line 34WA009 and to watermelonplants, parts thereof and seed produced using those methods. The presentinvention further relates to hybrid watermelon seeds and plants (andparts thereof including fruit) produced by crossing watermelon line34WA009 with another watermelon plant, e.g., hybrid triploid seeds andplants produced by crossing with a diploid plant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of watermelon line 34WA009. In embodiments, thetissue culture is capable of regenerating plants having all oressentially all of the physiological and morphological characteristicsof the foregoing watermelon plant and/or of regenerating plants havingthe same or substantially the same genotype as the foregoing watermelonplant. In embodiments, the regenerated plant is a tetraploid plant. Inexemplary embodiments, the regenerable cells in such tissue cultures aremeristematic cells, cotyledons, hypocotyl, leaves, pollen, embryos,roots, root tips, anthers, pistils, ovules, shoots, stems, petiole,pith, flowers, capsules, rootstock, scion and/or seeds as well as callusand/or protoplasts derived from any of the foregoing. Still further, thepresent invention provides watermelon plants regenerated from the tissuecultures of the invention.

As a further aspect, the invention provides a method of producingwatermelon seed, the method comprising crossing a plant of watermelonline 34WA009 with itself or a second watermelon plant and allowing seedto form (e.g., tetraploid or triploid hybrid seed). Optionally, themethod further comprises collecting the seed.

Another aspect of the invention provides methods for producing hybridsand other watermelon plants derived from watermelon line 34WA009.Watermelon plants derived by use of these methods are also part of theinvention as well as plant parts, seed, gametes and tissue culture fromsuch hybrid or derived watermelon plants.

In representative embodiments, a watermelon plant derived fromwatermelon line 34WA009 comprises cells comprising at least one set ofchromosomes derived from watermelon line 34WA009. In embodiments, thederived watermelon plant is a tetraploid plant. In embodiments, thederived watermelon plant is a triploid plant. In embodiments, thederived watermelon plant is a diploid plant.

In embodiments, a watermelon plant or population of watermelon plantsderived from watermelon line 34WA009 comprises, on average, at least6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of its alleles (i.e., theoreticalallelic content; TAC) from watermelon line 34WA009, e.g., at least about6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of the genetic complement ofwatermelon line 34WA009, and optionally may be the result of a breedingprocess comprising one or two breeding crosses and one or more ofselfing, sibbing, backcrossing and/or double haploid techniques in anycombination and any order. In embodiments, the breeding process does notinclude a breeding cross, and comprises selfing, sibbing, backcrossingand or double haploid technology. In embodiments, the derived watermelonplant is a tetraploid plant. In embodiments, the derived watermelonplant is a triploid plant. In embodiments, the derived watermelon plantis a diploid plant. In embodiments, the watermelon plant derived fromwatermelon line 34WA009 is one, two, three, four, five or more breedingcrosses removed from watermelon line 34WA009.

In embodiments, a hybrid or derived plant from watermelon line 34WA009comprises a desired added trait(s). In representative embodiments, awatermelon plant derived from watermelon line 34WA009 comprises all ofthe morphological and physiological characteristics of watermelon line34WA009 (e.g., as described in Tables 1 to 3). In embodiments, thewatermelon plant derived from watermelon line 34WA009 comprisesessentially all of the morphological and physiological characteristicsof watermelon line 34WA009 (as described herein), with the addition of adesired added trait(s). In embodiments, the plant derived from line34WA009 is a tetraploid plant. In embodiments, the plant derived fromline 34WA009 is a triploid plant. In embodiments, the plant derived fromline 34WA009 is a diploid plant.

The invention also relates to methods for producing a watermelon plantcomprising in its genetic material one or more transgenes and to thetransgenic watermelon plant produced by those methods (and progenywatermelon plants comprising the transgene). Also provided are plantparts, seed and tissue culture from such transgenic watermelon plants,optionally wherein one or more cells in the plant part, seed, or tissueculture comprises the transgene. The transgene can be introduced viaplant transformation and/or breeding techniques.

In another aspect, the present invention provides for single locusconverted plants (e.g., diploid, triploid or tetraploid) of watermelonline 34WA009. Plant parts, seed, and tissue culture from such singlelocus converted plants are also contemplated by the present invention.The single locus may be a dominant or recessive allele. Inrepresentative embodiments, the single locus confers such traits as malesterility, herbicide resistance, pest resistance (e.g., insect and/ornematode resistance), modified fatty acid metabolism, modifiedcarbohydrate metabolism, disease resistance (e.g., for bacterial, fungaland/or viral disease), male fertility, enhanced nutritional quality,improved appearance (e.g., color), improved salt tolerance, industrialusage, or any combination thereof. The single locus may be a naturallyoccurring watermelon locus, a genome edited locus, a mutated locus(e.g., chemically or radiation induced), or a transgene introduced intowatermelon through genetic engineering techniques.

The invention further provides methods for developing watermelon plants(e.g., diploid, triploid or tetraploid) in a watermelon plant breedingprogram using plant breeding techniques including, for example,recurrent selection, backcrossing, pedigree breeding, double haploidtechniques, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection and/or transformation. Seeds,watermelon plants, and parts thereof, produced by such breeding methodsare also part of the invention.

The invention also provides methods of multiplication or propagation ofwatermelon plants of the invention, which can be accomplished using anymethod known in the art, for example, via vegetative propagation and/orseed.

Additional aspects of the invention include harvested products andprocessed products from the watermelon plants of the invention. Aharvested product can be a whole plant or any plant part, as describedherein. Thus, in some embodiments, a non-limiting example of a harvestedproduct includes a seed, a fruit (e.g., including the flesh and/orrind), a rootstock, a scion and/or a shoot.

In representative embodiments, a processed product includes, but is notlimited to: cut, sliced, ground, pureed, dried, canned, jarred, washed,packaged, frozen and/or heated fruit (including the fruit flesh and/orrind) of the watermelon plants of the invention, or any other partthereof. In embodiments, the processed product includes washed andsliced fruit (or parts thereof, e.g., the fruit flesh with or withoutthe rind) of the invention.

The seed of the invention can optionally be provided as an essentiallyhomogenous population of seed of a single plant or cultivar. Essentiallyhomogenous populations of seed are generally free from substantialnumbers of other seed, e.g., at least about 90%, 95%, 96%, 97%, 98% or99% pure.

In representative embodiments, the invention provides a seed ofwatermelon line 34WA009.

As a further aspect, the invention provides a plant of watermelon line34WA009.

As an additional aspect, the invention provides a watermelon plant, or apart thereof, having all or essentially all of the physiological andmorphological characteristics of a plant of watermelon line 34WA009.Optionally, the plant having all or essentially all of the physiologicaland morphological characteristics of a plant of watermelon line 34WA009is a tetraploid plant.

The invention also provides plants that are diploid reversions oftetraploid watermelon line 34WA009 and parts (including seed and fruits)thereof. Also provided are methods of producing a diploid reversionderived from watermelon line 34WA009.

Also provided are methods of producing a diploid reversion of tetraploidwatermelon plant of the invention. In embodiments, the method comprisescrossing two plants of watermelon line 34WA009 or selfing watermelonline 34WA009, harvesting seed, and growing a diploid reversion from theharvested seed. In embodiments, the fruit and/or seed of the diploidreversion is collected. Optionally, the diploid reversion can be used ina watermelon breeding program. For example, the diploid reversion can becrossed with itself or another diploid watermelon plant to produce afurther watermelon plant, fruit and seed derived from watermelon line34WA009. In embodiments, the method further comprises doubling thechromosomes of the diploid reversion (or a diploid watermelon progenythereof) to produce a tetraploid watermelon plant.

The invention further provides triploid watermelon seed and triploidwatermelon plants (and parts thereof, such as seedless fruit) producedby crossing watermelon line 34WA009 with a diploid plant. Optionally,34WA009 is used as the female parent.

As another aspect, the invention provides fruit and/or seed of thewatermelon plants of the invention and a processed product from thefruit and/or seed of the inventive watermelon plants.

As still another aspect, the invention provides a method of producingwatermelon seed, the method comprising crossing a watermelon plant ofthe invention with itself or a second watermelon plant. In embodiments,the method is practiced to produce seed of line 34WA009 (e.g., seedincrease) by crossing the plant with itself, e.g., by open pollinationof the variety. The invention also provides seed produced by this methodand plants, and parts thereof including fruit, produced by growing theseed.

As yet a further aspect, the invention provides a method for producing aseed of a watermelon plant derived from watermelon line 34WA009, themethod comprising: (a) crossing a watermelon plant of watermelon line34WA009 with a second watermelon plant; and (b) allowing seed of awatermelon plant derived from watermelon line 34WA009 to form. Inembodiments, the method further comprises: (c) growing a plant from theseed derived from watermelon line 34WA009 of step (b); (d) selfing theplant of step (c) or crossing it to a second watermelon plant to formadditional watermelon seed derived from watermelon line 34WA009, and (e)optionally repeating steps (c) and (d) one or more times to generatefurther derived watermelon seed from watermelon line 34WA009, wherein instep (c) a plant is grown from the additional watermelon seed of step(d) in place of growing a plant from the seed of step (b). Inembodiments, the method comprises: (e) repeating steps (c) and (d) oneor more times (e.g., one to three, one to five, one to six, one toseven, one to ten, three to five, three to six, three to seven, three toeight or three to ten times) to generate further derived watermelonseed. As another option, the method can comprise collecting the seed.The invention also provides seed produced by these methods andwatermelon plants (e.g., tetraploid watermelon plants) produced bygrowing the seed.

Still further, as another aspect, the invention provides a method ofvegetatively propagating a plant of watermelon line 34WA009, e.g., viashoot proliferation and then rooting in tissue culture. Detailed methodswere described by Zhang et al. (Zhang, X. P., B. B. Rhodes, H. T.Skorupska, W. C. Bridges. 1995. Generating Tetraploid Watermelon UsingColchicine in Vitro. G. Lester & J. Dunlap et al. (eds.), Cucurbitaceae'94: 134-139). In a non-limiting example, the method comprises: (a)collecting tissue capable of being propagated from a plant of watermelonline 34WA009; (b) cultivating the tissue to obtain proliferated shoots;and (c) rooting the proliferated shoots to obtain rooted plantlets.Optionally, the invention further comprises growing plants from therooted plantlets. The invention also encompasses the plantlets andplants produced by these methods.

As an additional aspect, the invention provides a method of introducinga desired added trait into watermelon line 34WA009, the methodcomprising: (a) crossing a first plant of watermelon line 34WA009 with asecond watermelon plant that comprises a desired trait to produce F₁progeny; (b) selecting an F₁ progeny that comprises the desired trait;(c) crossing the selected F₁ progeny with watermelon line 34WA009 toproduce backcross progeny; and (d) selecting backcross progenycomprising the desired trait to produce a plant derived from watermelonline 34WA009 comprising a desired trait.

In embodiments, the selected progeny produces a fruit with thecharacteristics of line 34WA009 previously mentioned above. Inembodiments, the selected progeny comprises all or essentially all themorphological and physiological characteristics of watermelon line34WA009 (e.g., a described in Tables 1 to 3). Optionally, the methodfurther comprises: (e) repeating steps (c) and (d) one or more times(e.g., one to three, one to five, one to six, one to seven, one to ten,three to five, three to six, three to seven, three to eight or three toten times) to produce a plant derived from watermelon line 34WA009comprising the desired trait, wherein in step (c) the selected backcrossprogeny produced in step (d) is used in place of the selected F1 progenyof step (b). In embodiments, the plant derived from line 34WA009 andcomprising the desired added trait is a tetraploid plant. Inembodiments, the plant derived from line 34WA009 and comprising thedesired added trait is a triploid plant. In embodiments, the plantderived from line 34WA009 and comprising the desired added trait is adiploid plant.

In representative embodiments, the invention also provides a method ofproducing a plant of watermelon line 34WA009 comprising a desired addedtrait, the method comprising introducing a transgene conferring thedesired trait into a plant of watermelon line 34WA009. The transgene canbe introduced by transformation methods (e.g., genetic engineering) orbreeding techniques. In embodiments, the plant comprising the transgeneproduces a mature fruit with the characteristics of line 34WA009previously mentioned above. In embodiments, the plant comprising thetransgene comprises all or essentially all morphological andphysiological characteristics of watermelon line 34WA009 (e.g., asdescribed in Tables 1 to 3).

The invention also provides watermelon plants (e.g., a diploid, triploidor tetraploid watermelon plant) produced by the methods of theinvention, wherein the watermelon plant has the desired added trait aswell as seed and fruits from such watermelon plants. The invention alsoprovides seed that produces the plants derived from line 34WA009 andcomprising a desired added trait.

According to the foregoing methods, the desired added trait can be anysuitable trait known in the art including, for example, male sterility,male fertility, herbicide resistance, insect or pest (e.g., insectand/or nematode) resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, disease resistance (e.g., for bacterial, fungaland/or viral disease), enhanced nutritional quality, increasedsweetness, increased flavor, improved ripening control, improved salttolerance, industrial usage, or any combination thereof.

In representative embodiments, a transgene conferring herbicideresistance confers resistance to glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,L-phosphinothricin, cyclohexone, cyclohexanedione, triazine,benzonitrile, or any combination thereof.

In representative embodiments, a transgene conferring pest resistance(e.g., insect and/or nematode resistance) encodes a Bacillusthuringiensis endotoxin.

In representative embodiments, a watermelon plant of the invention(including transgenic plants, single locus converted plants, hybridplants and watermelon plants derived from watermelon line 34WA009, andplants produced by the methods of the invention) are characterized by,e.g., one or more of tetraploidy, round fruits, mature fruits having astriped pattern (e.g., Crimson Sweet) skin color, an intermediate rindthickness, an intense red flesh (e.g., darker red as compared with line4XCS34), and/or resistance to Fusarium wilt race 1, Powdery Mildew race1, and/or Anthracnose race 1. In representative embodiments, transgenicplants, single locus converted plants, hybrid plants and watermelonplants derived from watermelon line 34WA009 have at least 3, 4, 5, 6, 7,8, 9, 10 or more of the morphological and physiological characteristicsof watermelon line 34WA009 mentioned above (e.g., as described in Tables1 to 3), or even all of the morphological and physiologicalcharacteristics of watermelon line 34WA009, so that said plants are notsignificantly different for said traits than watermelon line 34WA009, asdetermined at the 5% significance level when grown in the sameenvironmental conditions; optionally, with the presence of one or moredesired additional traits (e.g., male sterility, disease resistance,pest or insect resistance, herbicide resistance, and the like).

As a further aspect, the invention provides a method of producingtriploid watermelon seed, the method comprising: (a) crossing awatermelon plant of line 34WA009 with a diploid watermelon plant; and(b) optionally, harvesting the resultant triploid watermelon seed. Inembodiments, the plant of line 34WA009 is the female parent and thediploid parent is the male plant (i.e., pollinator). Also provided is ahybrid triploid watermelon seed produced by the foregoing method, and atriploid watermelon plant (and parts thereof, including seedless fruit)grown from the triploid seed.

The invention also provides as another aspect, a method of producingseedless watermelon fruit, the method comprising: (a) crossing thetriploid watermelon plant produced by the method of the precedingparagraph and a diploid watermelon plant; (b) allowing a seedless fruitto form; and (c) optionally, harvesting the seedless fruit. Inembodiments, the plant of line 34WA009 is the female parent and thediploid parent is the male plant (i.e., pollinator). Also provided is aseedless watermelon fruit produced by the foregoing method.

As still a further aspect, the invention provides a tetraploidwatermelon plant, or a part thereof, produced by crossing a 34WA009plant with a different tetraploid watermelon plant (e.g., a tetraploidinbred or hybrid plant).

The invention further provides a method of developing a tetraploidwatermelon line in a watermelon plant breeding program using plantbreeding techniques, which include employing a watermelon plant, or apart thereof, as a source of plant breeding material, the methodcomprising: (a) obtaining the watermelon plant, or a part thereof, ofline 34WA009 as a source of breeding material; and (b) applying plantbreeding techniques.

The invention also encompasses plant parts, plant material, pollen,ovules, leaves, fruit and seed from the watermelon plants of theinvention. The invention also provides seed that produces the watermelonplants of the invention. Also provided is a tissue culture ofregenerable cells from the watermelon plants of the invention, whereoptionally, the regenerable cells are: (a) embryos, meristem, leaves,pollen, cotyledons, hypocotyls, roots, root tips, anthers, flowers,pistils, ovules, seed, shoots, stems, stalks, petioles, pith and/orcapsules; or (b) callus or protoplasts derived from the cells of (a).Further provided are watermelon plants regenerated from a tissue cultureof the invention.

In still yet another aspect, the invention provides a method ofdetermining a genetic characteristic of watermelon line 34WA009 or aprogeny thereof, e.g., a method of determining a genotype of watermelonline 34WA009 or a progeny thereof using molecular genetic techniques. Inembodiments, the method comprises detecting in the genome of a 34WA009plant, or a progeny plant thereof, at least a first polymorphism, e.g.,comprises nucleic acid amplification and/or nucleic acid sequencing. Toillustrate, in embodiments, the method comprises obtaining a sample ofnucleic acids from the plant and detecting at least a first polymorphismin the nucleic acid sample (e.g., using one or more molecular markers).Optionally, the method may comprise detecting a plurality ofpolymorphisms (e.g., two or more, three or more, four or more, five ormore, six or more, eight or more or ten or more polymorphisms, etc.) inthe genome of the plant. In representative embodiments, the methodfurther comprises storing the results of the step of detecting thepolymorphism(s) on a computer readable medium. The invention furtherprovides a computer readable medium produced by such a method.

In addition to the exemplary aspects and embodiments described above,the invention is described in more detail in the description of theinvention set forth below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the development of a novelwatermelon characterized by producing mature fruits having a roundshape, a striped pattern (Crimson Sweet) skin color, intermediate rindthickness, an intense red flesh color, and having resistance to FusariumWilt race 1, Powdery Mildew race 1, and Anthracnose race 1. As comparedwith tetraploid line 4XCS34, the mature fruits of line 34WA009 have agreater maximum fruit weight, are uniform in shape, and have a darkerred flesh color.

It should be appreciated that the invention can be embodied in differentforms and should not be construed as limited to the embodiments setforth herein. Rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

Unless the context indicates otherwise, it is specifically intended thatthe various features and embodiments of the invention described hereincan be used in any combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. To illustrate, if thespecification states that a composition comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Definitions

In the description and table that follow, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463(CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus,the term “consisting essentially of” when used in a claim or thedescription of this invention is not intended to be interpreted to beequivalent to “comprising.”

Those skilled in the art will appreciate that when a comparison ofphysiological and morphological characteristics between two or morevarieties is made, it is assumed that the varieties are grown under thesame environmental conditions, whether in the field or green house. Inaddition, such comparisons are generally made on the basis ofobservations taken on a population of plants, which define thecharacteristics of the variety.

“Allele”. An allele is any of one or more alternative forms of a gene,all of which relate to a trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

“Backcrossing”. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

“Cotyledon”. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

“Double haploid line”. A stable inbred line achieved by doubling thechromosomes of a haploid line, e.g., from anther culture. For example,some pollen grains (haploid) cultivated under specific conditionsdevelop plantlets containing 1 n chromosomes. The chromosomes in theseplantlets are then induced to “double” (e.g., using chemical means)resulting in cells containing 2n chromosomes. The progeny of theseplantlets are termed “double haploid” and are essentially notsegregating any more (e.g., are stable). The term “double haploid” isused interchangeably herein with “dihaploid.”

“Essentially all the physiological and morphological characteristics”. Aplant having “essentially all the physiological and morphologicalcharacteristics” (and similar phrases) means a plant having all of thedesired physiological and morphological characteristics of variety34WA009, except for the characteristic(s) derived from a convertedlocus/loci (e.g., a single converted locus), for example, introduced viabackcrossing to variety 34WA009, a modified gene(s) resulting fromgenome editing techniques, an introduced transgene (i.e., introduced viagenetic transformation techniques) or mutation, when both plants aregrown under the same environmental conditions. In embodiments, a planthaving “essentially all of the physiological and morphologicalcharacteristics” means a plant having all of the characteristics of thereference plant with the exception of five or fewer traits, 4 or fewertraits, 3 or fewer traits, 2 or fewer traits, or one trait. Inembodiments, the plant comprising “essentially all of the physiologicaland morphological characteristics” of variety 34WA009 produces a maturefruit with a round shape, a striped pattern (Crimson Sweet) skin color,intermediate rind thickness, and a dark red flesh color (e.g., darkerred as compared with tetraploid line 4XCS34) and has resistance toFusarium Wilt race 1, Powdery mildew race 1, and Anthracnose race 1, andis optionally diploid or tetraploid. In embodiments, a plant comprising“essentially all of the physiological and morphological characteristics”of variety 34WA009 comprises the traits of Tables 1 to 3.

“First water date”. The date the seed first receives adequate moistureto germinate. This can and often does equal the planting date.

“Gene”. As used herein, “gene” refers to a segment of nucleic acidcomprising an open reading frame. A gene can be introduced into a genomeof a species, whether from a different species or from the same species,using transformation or various breeding methods.

“Inbred line”: As used herein, the phrase “inbred line” refers to agenetically homozygous or nearly homozygous population. An inbred line,for example, can be derived through several cycles of sib crossingand/or selfing and/or via double haploid production. In someembodiments, inbred lines breed true for one or more traits of interest.An “inbred plant” or “inbred progeny” is an individual sampled from aninbred line.

“Plant.” As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, fruit, stems, rootstock, scion, and the like.

“Plant material”. The terms “plant material” and “material obtainablefrom a plant” are used interchangeably herein and refer to any plantmaterial obtainable from a plant including without limitation, leaves,stems, roots, flowers or flower parts, fruits, pollen, ovules, zygotes,seeds, cuttings, cell or tissue cultures, rootstocks, scions, or anyother part or product of the plant.

“Plant part”. As used herein, a “plant part” includes any part, organ,tissue or cell of a plant including without limitation an embryo,meristem, leaf, pollen, cotyledon, hypocotyl, root, root tip, anther,flower, flower bud, pistil, ovule, seed, shoot, stem, stalk, petiole,pith, capsule, a scion, a rootstock and/or a fruit including callus andprotoplasts derived from any of the foregoing. In representativeembodiments, the plant part is a non-propagating plant part, forexample, is not a seed.

“Quantitative Trait Loci”. Quantitative Trait Loci (QTL) refers togenetic loci that control to some degree, numerically representabletraits that are usually continuously distributed.

“Regeneration”. Regeneration refers to the development of a plant fromtissue culture.

“Resistance”. As used herein the terms “resistance” and “tolerance” (andgrammatical variations thereof) are used interchangeably to describeplants that show reduced or essentially no symptoms to a specific biotic(e.g., a pest, pathogen or disease) or abiotic (e.g., exogenous orenvironmental, including herbicides) factor or stressor. In someembodiments, “resistant” or “tolerant” plants show some symptoms but arestill able to produce marketable product with an acceptable yield, e.g.,the yield may still be reduced and/or the plants may be stunted ascompared with the yield or growth in the absence of the biotic and/orabiotic factor or stressor. Those skilled in the art will appreciatethat the degree of resistance or tolerance may be assessed with respectto a plurality or even an entire field of plants. A watermelon plant maybe considered “resistant” or “tolerant” if resistance/tolerance isobserved over a plurality of plants (e.g., an average), even ifparticular individual plants may be susceptible to the biotic or abioticfactor or stressor.

“RHS”. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

“Single locus converted”. A single locus converted or conversion plantrefers to a plant that is developed by plant breeding techniques (e.g.,backcrossing), genome editing techniques, genetic transformationtechniques and/or mutation techniques wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single locus introduced into the line viathe plant breeding, genome editing, genetic transformation, or mutationtechniques.

As used herein, a “small” watermelon fruit refers to a mean fruit weightthat is less than about 8 kg, 7 kg, 6 kg, 5 kg, 4.5 kg or even 4 kg.

“Substantially equivalent characteristic”. A characteristic that, whencompared, does not show a statistically significant difference (e.g.,p=0.05) from the mean.

“Thousand seed weight” as used herein refers to the average weight of1000 seeds of the variety. In embodiments, the plant or variety has a“low” or “relatively low” thousand seed weight, e.g., less than about55, 50, 45, 44, 43, 42, 41 or 40 grams per thousand seeds (e.g.,untreated seeds).

“Transgene”. A nucleic acid of interest that can be introduced into thegenome of a plant by genetic engineering techniques (e.g.,transformation) or breeding. The transgene can be from the same or adifferent species. If from the same species, the transgene can be anadditional copy of a native coding sequence or can present the nativesequence in a form or context (e.g., different genomic location and/orin operable association with exogenous regulatory elements such as apromoter) than is found in the native state. The transgene can comprisean open reading frame encoding a polypeptide or can encode a functionalnon-translated RNA (e.g., RNAi).

Botanical Description of the Watermelon Line 34WA009.

Characteristics. New watermelon inbred tetraploid line 34WA009 ischaracterized by producing mature fruits having a round shape, a stripedpattern (Crimson Sweet) skin color, intermediate rind thickness, and anintense red flesh color, and resistance to Fusarium Wilt race 1, Powderymildew race 1, and Anthracnose race 1. As compared with tetraploid line4XCS34 (United States PVPO No. 201000271), it has higher plant vigor, agreater maximum fruit weight, and a darker red flesh color. Thiscombination of traits is unique to 34WA009. Line 4XCS34 was selected asthe most similar variety for comparison because it is a tetraploid linewith intermediate resistance to Fusarium wilt race 1 and has a similarfruit shape, size, and rind color as compared with line 34WA009.

Promising triploid hybrids produced using 34WA009 as female parent havebeen developed. 34WA009 is a new, unique and useful elite inbredtetraploid line for producing triploid seedless hybrids.

Watermelon line 34WA009 has shown uniformity and stability within thelimits of environmental influence. It has been self-pollinated fornumerous generations with careful attention to uniformity of plant type.The variety has been increased with continued observation foruniformity. No variant traits have been observed or are expected inwatermelon line 34WA009.

A more detailed botanical description of 34WA009 and comparison with4XCS34 is shown in Table 1 below.

TABLE 1 Description of 34WA009 and comparison with 4XCS34 based on openfield trials in Naples, Florida. Characteristic 34WA009 4XCS34 Generalfruit type: Round large Round large Area of best Most U.S. areas MostU.S. areas adaptation: Maturity -No. of days 24 days 24 days fromemergence to anthesis: Maturity -No. of days 44 days 42 days frompollination to maturity: Maturity -days Relative 79 days 75 daysMaturity: Maturity category: Medium Medium Ploidy: Tetraploid TetraploidCotyledon shape: Flat Flat Plant sex form: Monoecious Monoecious Numberof flowers per 3.45 staminate, 0.4 2.65 staminate, 0.1 plant at firstfruit set: pistillate, 0 perfect pistillate, 0 perfect Stem shape: Round(cross-section) Round (cross-section) Stem surface: Pubescent PubescentVine length at last 112.7 cm 108.7 cm harvest: No. internodes at last24   31   harvest: Ratio: cm vine  4.69  3.49 length/internodes at lastharvest: Leaf shape: Ovate Ovate Leaf lobes: Lobed Lobed Leaf length:16.405 cm 19.415 cm Leaf width: 18.435 cm 20.71 cm Leaf size: Wider thanlong Wider than long Leaf dorsal surface Smooth Smooth pubescence: Leafventral surface Smooth Smooth pubescence: Leaf color: Medium greenMedium green Flower (staminate 3.7 cm across 3.2 cm across diameter):Flower (Pistillate 2 cm across 2 cm across diameter): Flower (Perfectn/a n/a diameter): Flower color: Yellow Yellow Mature fruit shape: RoundRound Mature fruit length: 21.6 cm 22.34 cm Mature fruit diameter 20.69cm 22.46 cm (midsection): Mature fruit average 5.48 kg 6.15 kg weight:Mature fruit maximum 7.4 kg 7.2 kg weight: Mature fruit index = 423  448   length ÷ diameter × 10: Mature fruit surface: Smooth Smooth Maturefruit skin color Stripe Stripe pattern: Mature fruit primary Light GreenLight Green color: (Charleston Grey) (Charleston Grey) Mature fruitsecondary Medium green Medium green color: Rind texture: Tough ToughRing thickness blossom n/a n/a end: Rind thickness sides: 19 mm 15 mmFlesh texture: Crisp Crisp Flesh coarseness: Fine - little fiber Fine -little fiber Flesh color: Dark red Red Flesh - refractometer 9.5% 9.75%   (% soluble solids of juice, center of fruit): Flesh - % hollowheart: 10% 0% Flesh - % placental 10% 0% separation: Flesh - %transverse  0% 0% crack: Seed size (F1 seed - Medium Medium no seed inF1 fruit): Seed length (F1 seed - 9.3 mm 9.13 mm no seed in F1 fruit):Seed width (F1 seed - 6.5 mm 6.07 mm no seed in F1 fruit): Seedthickness (F1 2.6 mm 2 mm seed - no seed in F1 fruit): Seed index - 14.314.6 length ÷ width × 10 (F1 seed - no seed in F1 fruit): No. seeds perfruit (F1 90.3 76.4 seed - no seed in F1 fruit): Seed color (F1 seed -Dark brown mottled Tan no seed in F1 fruit): Fusarium Wilt, Race 1:Resistant Resistant Anthracnose, Race 1: Resistant Resistant

34WA009 has resistance to Fusarium wilt race 1, conferred by the geneFo-1 (Henderson, W. R., S. F. Jenkins, Jr., and J. O. Rawlings. 1970.The inheritance of Fusarium wilt resistance in watermelon, Citrulluslanatus (Thunb.) Mansf. J. Amer. Soc. Hort. Sci. 95: 276-282), asdemonstrated in the standard pathology test results shown in Table 2below.

TABLE 2 Test of Resistance to Fusarium wilt race 1, Powdery mildew race1, and Anthracnose race 1 in 34WA009 and controls. Aggregated results oftwo tests for resistance to Fusarium wilt race 1 in 34WA009 andcontrols. Variety Total Plants Inoculated R S %R 34WA009 53 51 0 96.2264Black Diamond 55 3 48 0.054 SP-6 56 51 1 91.0714 Standard Fusarium wiltresistance best protocol was used for these tests. Tests were conductedin the pathology greenhouse of the Woodland (California) station in theFall of 2018. “R” = resistant plants, “S” = susceptible plants.Aggregated results of five tests for resistance to Powdery Mildew race 1in 34WA009 and controls. Variety Total Plants Inoculated IR S %R 34WA009Total 40 38 2 95.0 BLACK DIAMOND 45 38 6 84.44 SP-6 Total 48 26 0 Testswere conducted in the pathology greenhouse of the Woodland (California)station in the Fall of 2018. “R” = resistant plants, “S” = susceptibleplants. Aggregated results of three tests for resistance to Anthracnoserace 1 in 34WA009 and controls. Variety Total Plants Inoculated R S %R34WA009 50 50 0 100 BLACK DIAMOND 55 2 53 0.03 SP-6 56 56 0 100 StandardAnthracnose resistance best protocol was used for these tests. Testswere conducted in the pathology greenhouse of the Woodland (California)station in the Fall of 2018. “R” = resistant plants, “S” = susceptibleplants.

34WA009 possesses unique aspects of fruit quality. When compared withline 4XCS34, 34WA009 has qualitatively darker red flesh than 4XCS34.34WA009 has large fruit size, high plant vigor, dark red flesh, highnumber of seed produced per fruit, and disease resistances as describedabove. This combination of traits is unique in 34WA009.

TABLE 3 Comparison of rind thickness, flesh firmness, and seed per fruitcharacteristics of line 34WA009 and 4XCS34 from Summer 2019 season inWoodland. Five fruit each were measured; the average of threereplications of 4XCS34 is used for comparison to 34WA009. Line Rindthickness (cm) Flesh firmness (kgf) Seed per fruit 4XCS34 1.5 1.85 7134WA009 1.63 1.38 71

Tissue Culture.

In embodiments, watermelon plants can be propagated by tissue cultureand regeneration. Tissue culture of various plant tissues andregeneration of plants therefrom is well known. For example, referencemay be had to Teng, et al., HortScience, 27:9, 1030-1032 (1992); Teng,et al., HortScience, 28:6, 669-1671 (1993); Zhang, et al., Journal ofGenetics and Breeding, 46:3, 287-290 (1992); Webb, et al., Plant CellTissue and Organ Culture, 38:1, 77-79 (1994); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Nagata, et al., Journalfor the American Society for Horticultural Science, 125:6, 669-672(2000); and Ibrahim, et al., Plant Cell Tissue and Organ Culture, 28(2),139-145 (1992). It is clear from the literature that the state of theart is such that these methods of obtaining plants are routinely usedand have a high rate of success. Thus, another aspect of this inventionis to provide cells which upon growth and differentiation producewatermelon plants having desired characteristics of watermelon line34WA009 (i.e., one or more of the characteristics previously listed ofthe fruit of line 34WA009.). Optionally, watermelon plants can beregenerated from the tissue culture of the invention comprising all oressentially all of the physiological and morphological characteristicsof watermelon line 34WA009.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques.

Tetraploid Watermelon Lines and Triploid Seed Production

The primary use of tetraploid watermelons is to make triploid hybridwatermelon seeds and plants that produce seedless fruit. In commercialproduction of triploid watermelon seed, tetraploid and diploid parentallines are typically planted in the same field. Cross-pollination betweenthe tetraploid line, generally used as the female parental line, and thediploid line, the male parental line, are accomplished by either hand orbee pollination. Triploid watermelon seeds are produced only in fruitsof tetraploid plants that are fertilized with pollen of diploid plants.All commercially grown seeded watermelons are diploid; therefore, thereare many diploid lines for use as diploid parents. The major limitationto development of seedless watermelon varieties lies in the availabilityof useful elite tetraploid parental lines.

Tetraploid watermelon lines can be developed from diploid lines bydoubling the chromosomes of diploid watermelon lines using methodsroutine in the art. Chromosome doubling was first accomplished with thealkaloid colchicine by applying colchicine to the growing point of newlyemerged watermelon seedlings. Tissue culture methods have also beendeveloped (Zhang, X. P., B. B. Rhodes, H. T. Skorupska, W. C. Bridges,1995, Generating Tetraploid Watermelon Using Colchicine in Vitro, G.Lester & J. Dunlap et al. (eds.), Cucurbitaceae' 94: 134-139).Dinitroanilines have been used to double chromosome numbers, and theireffectiveness has previously been compared with crops other thanwatermelon. Li et al. compared in vitro chromosome doublingeffectiveness using colchicine and the dinitroanilines, ethalfluralin(N-ethyl-N-2-methyl-2-propenyl)-2,6-dinitro-4-(trifluoromethyl)benzanine), and oryzalin (3,5-dinitro-N4, N4-dipropylsulfanilamide) andconcluded that either ethalfluralin or oryzalin was preferable tocolchicine (Li, Ying, J. F. Whitesides, B. Rhodes, 1999, In vitrogeneration of tetraploid watermelon with two different dinitroanilinesand colchicines, Cucurbit Genetics Cooperative Rpt 22:38-40).

Several treatment methods can be used to induce tetraploids fromdiploids using the chemicals mentioned above. One exemplary method is totreat the seed before sowing. The seed are soaked in clean water for 5-6hrs and then the seed are soaked in either colchicine solution (0.2%) ordinitroanilines (e.g. 35 μM oryzalin) for 24 hrs. The seed are brieflyrinsed before sowing. Dry seed can also be directly soaked in thechemical solution without pre-soaking in the water. The treatmentusually reduces the germination and emergence. A second method is totreat the newly emerged seedling. To illustrate, the diploid inbreds canbe sown in the greenhouse in seedling flats. The soil temperature iskept at 29-31° C. for rapid and uniform germination. One drop ofcolchicine (0.1%) or dinitroanilines (e.g. 35 μM oryzalin) solution isadded to the shoot apex between the cotyledons as soon as the seedlinghas emerged from soil. The colchicine solution is applied to the growingpoint in the morning or evening for three consecutive days. Goodchromosome doubling is achieved from application of oryzalin. Anotherillustrative method is to treat the shoot apex of germinated seed afterwhich the germinated seed is planted into soil. The seeds are germinatedin an incubator at 30° C. When the radicals are about 2 cm long, theportion above the hypocotyls of germinated seeds is immersed upside downinto colchicine (0.1%) or dinitroaniline solution (35 μM oryzalin) for10-15 hrs at 30° C. in an incubator. The treatment is typicallyconducted in a high humidity chamber or box to assure that theradicals/roots are not desiccated. The seeds are then washed and plantedin the soil. The last two methods, although more tedious to use, usuallygive better recovery of tetraploid events as the root system is notaffected by the treatment.

The next step is to develop tetraploid lines from individual convertingevents. For example, the selected tetraploid individuals based onmorphological expression can be self-pollinated and the resulting seedsplanted in the next generation as lines. These lines can again beself-pollinated and compared for fertility and horticultural traits.Only the desirable lines are selected if there is difference among theselines. Desirable lines may be bulk harvested if there is no variationwithin the line and among selected lines. Further seed increases may beconducted in an isolation block. Mass selection may be conducted forthis increase in the isolation plot and thereafter. Fertility of thetetraploid may be improved in subsequent generations.

The use of tissue culture to propagate tetraploid watermelon plants isexemplified in Adelberg, J. W., B. B. Rhodes, Micropropagation fromzygotic tissue of watermelon, C. E. Thomas (ed.) Proc. of theCucurbitaceae 89: Evaluation and enhancement of cucurbit germplasm,Charleston S. C., USA; and Zhang et al., Shoot regeneration fromimmature cotyledon of watermelon, Cucurbit Genetics Coop. 17:111-115(1994).

Crossing two different tetraploids and then going through recombinationbreeding can also result in new tetraploid lines. A longer breedingperiod is typically employed to develop a stable tetraploid line usingthis approach because of the larger number of combinations and the fewerseed that tetraploids produce. However, some breeders have made goodprogress by taking this approach.

Because meiosis is sometimes irregular in autotetraploids, there can bediploids and aneuploids among the offspring. The leaves, flowers andpollen grains of tetraploids are morphologically distinct from diploids(Zhang, X. P., B. B. Rhodes, H. T. Skorupska, W. C. Bridges, 1995,Generating Tetraploid Watermelon Using Colchicine in Vitro, G. Lester &J. Dunlap et al. (eds.), Cucurbitaceae' 94: 134-139). Tetraploids alsohave a different number of chloroplasts in the guard cells (Compton, M.E., D. J. Gray and G. W. Elmstrom. 1996, Identification of tetraploidregenerants from cotyledons of diploid watermelon cultures in vitro,Euphytica 87:165-172). These morphological traits can help the breedereliminate the diploids and aneuploids occurring in the tetraploidpopulation during sexual propagation. Diploid reversions can also beidentified in situations in which a diploid derived from line 34WA009 isdesired, and such diploid reversions are also encompassed by the presentinvention.

Accordingly, the invention contemplates as one aspect a method ofproducing triploid watermelon seed, the method comprising: (a) crossingthe watermelon plant of line 34WA009 with a diploid watermelon plant;and (b) harvesting the resultant triploid watermelon seed. Inembodiments the plant of line 34WA009 is the female parent and thediploid plant is the male parent. In embodiments, the plant of line34WA009 is the male parent and the diploid plant is the female parent.The triploid watermelon seed produces a triploid plant, which when growninto a plant produces a seedless watermelon fruit (i.e., when crossedwith a diploid plant).

The invention further provides a method of producing seedless watermelonfruit, the method comprising: (a) crossing a triploid plant producedfrom line 34WA009 (e.g., an F1 hybrid of 34WA009 produced as describedin the preceding paragraph) and a diploid watermelon plant; (b) allowingseedless fruit to form; and (c) optionally, harvesting the seedlessfruit. In embodiments, the triploid watermelon seed and seed from adiploid plant are planted in one or more rows, and the plants areallowed to mature and develop seedless fruit. In embodiments, diploidand triploid seed are planted in the same row. In embodiments thetriploid plant is the female parent and the diploid plant is the maleparent. In embodiments, the triploid plant is the male parent and thediploid plant is the female parent.

Several methods can be used to produce triploid seeds from inbred34WA009. Two commonly used methods are described below. Variations tothese methods can be made according to the actual production situation.

Hand-Pollination Method

Hand pollination can be used for producing triploid seed from 34WA009.For example, in embodiments, the inbred tetraploid female parent 34WA009and the inbred diploid male parent line are planted in the same field.To illustrate, in an exemplary method, the inbred male parent is planted7-10 days earlier than the female parent 34WA009 to ensure adequatepollen supply at the pollination time. The male parent and female parent34WA009 can be planted, for example, in the ratio of 1 male parent to4-10 female parents. Optionally, the diploid male parent is planted atthe top of the field for efficient male flower collection duringpollination. Pollination is generally started when the second femaleflower on the tetraploid female parent 34WA009 is ready to flower.Female flower buds that are ready to open the next day are identified,covered with paper cups or small paper bags that prevent bee or anyother insect visit of the female flowers, and marked with any kind ofmaterial that can be easily seen the next morning. The male flowers ofthe diploid male parent are collected in the morning before they areopen and visited by pollinating insects. The covered female flowers ofthe tetraploid female parent, which have opened, are uncovered andpollinated with the collected fresh male flowers of the diploid maleparent, starting after the male flower sheds pollen. The pollinatedfemale flowers are again covered after pollination to prevent bees andany other insect visits. The pollinated female flowers are also marked.Generally, only the marked fruits are harvested for extracting triploidhybrid seed.

Bee-Pollination Method

Bee pollination can also be used in triploid watermelon production. Inan exemplary bee-pollination method, the tetraploid female parent34WA009 and the diploid male parent are typically planted in a ratio of2 rows tetraploid parent to 1 row male parent. The female tetraploidplants are pruned to 2-3 branches. All of the male flower buds on thefemale tetraploid parent plants are removed manually (the de-buddingprocess) during the pollination season, typically on a daily basis.Beehives are placed in the field for transfer of pollen by bees from themale parent to the female flowers of the female parent. Fruits setduring this de-budding time are marked. Generally, only the markedfruits are harvested for extracting hybrid triploid seed.

Additional Breeding Methods.

This invention is also directed to methods for producing a watermelonplant by crossing a first parent watermelon plant with a second parentwatermelon plant wherein the first or second parent watermelon plant isa plant of watermelon line 34WA009. Further, both first and secondparent watermelon can come from watermelon line 34WA009. Thus, any ofthe following exemplary methods using watermelon line 34WA009 are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, double haploid production, and the like. All plantsproduced using watermelon line 34WA009 as at least one parent are withinthe scope of this invention, including those developed from watermelonplants derived from watermelon line 34WA009. Advantageously, watermelonline 34WA009 can be used in crosses with other, different, watermelonplants to produce first generation (F₁) watermelon hybrid seeds andplants with desirable characteristics. The watermelon plants of theinvention can also be used for transformation where exogenous transgenesare introduced and expressed by the plants of the invention. Geneticvariants created either through traditional breeding methods or throughtransformation of the cultivars of the invention by any of a number ofprotocols known to those of skill in the art are intended to be withinthe scope of this invention.

The following describes exemplary breeding methods that may be used withwatermelon line 34WA009 in the development of further watermelon plants.One such embodiment is a method for developing watermelon line 34WA009progeny watermelon plants in a watermelon plant breeding programcomprising: obtaining a plant, or a part thereof, of watermelon line34WA009, utilizing said plant or plant part as a source of breedingmaterial, and selecting a watermelon line 34WA009 progeny plant withmolecular markers in common with watermelon line 34WA009 and/or withsome, all or essentially all morphological and/or physiologicalcharacteristics of watermelon line 34WA009 (see, e.g., Tables 1 to 3).In representative embodiments, the progeny plant has at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more of the morphological and physiologicalcharacteristics of watermelon line 34WA009 previously mentioned above oreven all of the morphological and physiological characteristics ofwatermelon line 34WA009 so that said progeny watermelon plant is notsignificantly different for said traits than watermelon line 34WA009, asdetermined at the 5% significance level when grown in the sameenvironmental conditions; optionally, with the presence of one or moredesired additional traits (e.g., male sterility, disease resistance,pest or insect resistance, herbicide resistance, and the like). Breedingsteps that may be used in the breeding program include pedigreebreeding, backcrossing, mutation breeding and/or recurrent selection. Inconjunction with these steps, techniques such as RFLP-enhancedselection, genetic marker enhanced selection (for example, SSR markers)and/or and the making of double haploids may be utilized.

Another representative method involves producing a population ofwatermelon line 34WA009 progeny plants, comprising crossing watermelonline 34WA009 with another watermelon plant, thereby producing apopulation of watermelon plants that, on average, derives at least6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of its alleles (i.e., TAC) fromwatermelon line 34WA009, e.g., at least about 6.25%, 12.5%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% of the genetic complement of watermelon line 34WA009. Oneembodiment of this invention is the watermelon plant produced by thismethod and that has obtained at least 6.25%, 12.5%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of its alleles (i.e., TAC) from watermelon line 34WA009, and optionallymay be the result of a breeding process comprising one or two breedingcrosses and one or more of selfing, sibbing, backcrossing and/or doublehaploid techniques in any combination and any order. In embodiments, thebreeding process does not include a breeding cross, and comprisesselfing, sibbing, backcrossing and or double haploid technology.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is oris not significant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus, the invention includes watermelonline 34WA009 progeny watermelon plants characterized by the traits of34WA009 previously mentioned In embodiments, the invention encompassesprogeny plants having a combination of at least 2, 3, 4, 5, 6, 7, 8, 9,10 or more of the characteristics as described herein for watermelonline 34WA009, so that said progeny watermelon plant is not significantlydifferent for said traits than watermelon line 34WA009, as determined atthe 5% significance level when grown in the same environmentalconditions. Using techniques described herein and those known in theart, molecular markers may be used to identify said progeny plant asprogeny of watermelon line 34WA009. Mean trait values may be used todetermine whether trait differences are significant, and optionally thetraits are measured on plants grown under the same environmentalconditions.

Progeny of watermelon line 34WA009 may also be characterized throughtheir filial relationship with watermelon line 34WA009, as for example,being within a certain number of breeding crosses of watermelon line34WA009. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross or a backcross to 34WA009 as a recurrent parent, made to selectamong existing genetic alleles. The lower the number of breeding crossesin the pedigree, the closer the relationship between watermelon line34WA009 and its progeny. For example, progeny produced by the methodsdescribed herein may be within 1, 2, 3, 4, 5 or more breeding crosses ofwatermelon line 34WA009.

In representative embodiments, a watermelon plant derived fromwatermelon line 34WA009 comprises cells comprising at least one set ofchromosomes derived from watermelon line 34WA009. In embodiments, thewatermelon plant or population of watermelon plants derived fromwatermelon line 34WA009 comprises, on average, at least 6.25%, 12.5%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% of its alleles (i.e., TAC) from watermelon line34WA009, e.g., at least about 6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of thegenetic complement of watermelon line 34WA009, and optionally may be theresult of one or more of selfing, sibbing, backcrossing and/or doublehaploid techniques in any combination. In embodiments, the watermelonplant derived from watermelon line 34WA009 is one, two, three, four,five or more breeding crosses removed from watermelon line 34WA009.

In representative embodiments, a plant derived from watermelon line34WA009 is a double haploid plant, a hybrid plant, an inbred plant, atetraploid plant, a triploid plant and/or a diploid plant.

In embodiments, a derived plant from watermelon line 34WA009 comprises adesired added trait. In representative embodiments, a watermelon plantderived from watermelon line 34WA009 comprises all of the morphologicaland physiological characteristics of watermelon line 34WA009 (asdescribed in Tables 1 to 3). In embodiments, the watermelon plantderived from watermelon line 34WA009 comprises essentially all of themorphological and physiological characteristics of watermelon line34WA009 (e.g., as described herein), with the addition of a desiredadded trait.

According to the invention, tetraploid inbreds can be used as parentallines to develop new tetraploid lines. The unique and desirable traitsof 34WA009 make it useful as a parental line in the development of newtetraploid inbreds. 34WA009 can be used as either female or male parentto cross with another tetraploid watermelon (e.g., and inbred or hybridtetraploid) to develop new tetraploid inbreds.

Those skilled in the art will appreciate that any of the traitsdescribed herein with respect to plant transformation methods can beintroduced into a plant of the invention (e.g., watermelon line 34WA009and hybrid watermelon plants and other watermelon plants derivedtherefrom) using breeding techniques.

Genetic Transformation.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign nucleic acids including additional or modified versions ofnative (endogenous) nucleic acids (optionally driven by a non-nativepromoter) in order to alter the traits of a plant in a specific manner.Any nucleic acid sequences, whether from a different species, the samespecies or an artificial sequence, which are introduced into the genomeusing transformation or various breeding methods, are referred to hereincollectively as “transgenes.” Over the last fifteen to twenty years,several methods for producing transgenic plants have been developed, andin particular embodiments the present invention also relates totransformed versions of watermelon plants disclosed herein.

Genetic engineering techniques can be used (alone or in combination withbreeding methods) to introduce one or more desired added traits intoplant, for example, watermelon 34WA009 or progeny or watermelon plantsderived thereof. Once a transgene has been introduction into a plant bygenetic transformation, it can be transferred to other plants viaconventional breeding.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. Optionally, such avector comprises one or more nucleic acids comprising a coding sequencefor a polypeptide or an untranslated functional RNA under control of, oroperatively linked to, a regulatory element (for example, a promoter).In representative embodiments, the vector(s) may be in the form of aplasmid and can be used alone or in combination with other plasmids, toprovide transformed watermelon plants using transformation methods asdescribed herein to incorporate transgenes into the genetic material ofthe watermelon plant.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct nucleic acid transfermethod, such as microprojectile-mediated delivery (e.g., with abiolistic device), DNA injection, Agrobacterium-mediated transformation,electroporation, and the like. Transformed plants obtained from theplants (and parts and tissue culture thereof) of the invention areintended to be within the scope of this invention.

Expression Vectors for Plant Transformation—Selectable markers.

Expression vectors typically include at least one nucleic acidcomprising or encoding a selectable marker, operably linked to aregulatory element (for example, a promoter) that allows transformedcells containing the marker to be either recovered by negativeselection, e.g., inhibiting growth of cells that do not contain theselectable marker, or by positive selection, e.g., screening for theproduct encoded by the selectable marker. Many commonly used selectablemarkers for plant transformation are well known in the transformationart, and include, for example, nucleic acids that code for enzymes thatmetabolically detoxify a selective chemical agent which may be anantibiotic or an herbicide, or nucleic acids that encode an alteredtarget which is insensitive to the inhibitor. Positive selection methodsare also known in the art.

Commonly used selectable markers in plants include, but are not limitedto: neomycin phosphotransferase II (nptII) conferring resistance tokanamycin, hygromycin phosphotransferase conferring resistance to theantibiotic hygromycin, bacterial selectable markers that conferresistance to antibiotics (e.g., gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, selectable markers conferring resistance to herbicides(e.g., glyphosate, glufosinate, or bromoxynil). Selection of transformedplant cells can also be based on screening presumptively transformedplant cells rather than direct genetic selection of transformed cellsfor resistance to a toxic substance such as an antibiotic; such markersinclude without limitation alpha-glucuronidase (GUS),alpha-galactosidase, luciferase, and Green Fluorescent Protein (GFP) andmutant GFPs.

Expression Vectors for Plant Transformation—Promoters.

Transgenes included in expression vectors are generally driven by anucleotide sequence comprising a regulatory element (for example, apromoter). Numerous types of promoters are well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells.

Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue-preferred.” Promoters thatinitiate transcription only in certain tissue are referred to as“tissue-specific.” A “cell type” specific promoter preferentially drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” promoter is a promoterthat is under environmental control. Examples of environmentalconditions that may affect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue-specific,tissue-preferred, cell type specific, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions.

Many suitable promoters are known in the art and can be selected andused to achieve the desired outcome.

Signal Sequences for Targeting Proteins to Subcellular Compartments.

Transport of polypeptides produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isgenerally accomplished by means of operably linking a nucleotidesequence encoding a signal sequence to the 5′ and/or 3′ region of anucleic acid encoding the polypeptide of interest. Signal sequences atthe 5′ and/or 3′ end of the coding sequence target the polypeptide toparticular subcellular compartments.

The presence of a signal sequence can direct a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J, 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Polypeptide Transgenes and Agronomic Transgenes.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign polypeptide then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981). According to a representative embodiment, the transgenic plantprovided for commercial production of foreign protein is a watermelonplant of the invention. In another embodiment, the biomass of interestis seed and/or fruit.

Likewise, by means of the present invention, agronomic transgenes andother desired added traits can be expressed in transformed plants (andtheir progeny, e.g., produced by breeding methods). More particularly,plants can be genetically engineered to express various phenotypes ofagronomic interest or other desired added traits. Exemplary nucleicacids of interest in this regard conferring a desired added trait(s)include, but are not limited to, those transgenes that confer resistanceto confer resistance to plant pests (e.g., nematode or insect) ordisease (e.g., fungal, bacterial or viral), transgenes that conferherbicide tolerance, transgenes that confer male sterility, andtransgenes that confer or contribute to a value-added trait such asincreased nutrient content (e.g., iron, nitrate), increased sweetness(e.g., by introducing a transgene coding for monellin), modified fattyacid metabolism (for example, by introducing into a plant an antisensesequence directed against stearyl-ACP desaturase to increase stearicacid content of the plant), modified carbohydrate composition (e.g., byintroducing into plants a transgene coding for an enzyme that alters thebranching pattern of starch), modified fruit color (e.g., external fruitcolor and/or fruit flesh), or modified flavor profile of the fruit.

In embodiments, the transgene encodes a non-translated RNA (e.g., RNAi)that is expressed to produce targeted inhibition of gene expression,thereby conferring the desired trait on the plant.

In embodiments, the transgene encodes the machinery used for geneediting techniques.

Any transgene, including those exemplified above, can be introduced intothe watermelon plants of the invention through a variety of meansincluding, but not limited to, transformation (e.g., genetic engineeringtechniques), conventional breeding, and introgression methods tointroduce the transgene into other genetic backgrounds.

Methods for Plant Transformation.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993). Commonly usedplant transformation methods include agrobacterium-mediatedtransformation and direct transgene transfer methods (e.g.,microprojectile-mediated transformation, sonication, liposome orspheroplast fusion, and electroporation of protoplasts or whole cells).

Following transformation of plant target tissues, expression ofselectable marker transgenes (e.g., as described above) allows forpreferential selection of transformed cells, tissues and/or plants,using regeneration and selection methods now well known in the art.

The foregoing methods for transformation are typically used to produce atransgenic watermelon line. The transgenic watermelon line can then becrossed with another (non-transgenic or transgenic) line in order toproduce a new transgenic watermelon line. Alternatively, a transgenethat has been engineered into a particular plant using transformationtechniques can be introduced into another plant or line usingtraditional breeding (e.g., backcrossing) techniques that are well knownin the plant breeding arts. For example, a backcrossing approach can beused to move an engineered transgene from a public, non-elite inbredline into an elite inbred line, or from an inbred line containing aforeign transgene in its genome into an inbred line or lines which donot contain that transgene. As used herein, “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

Locus Conversion.

When the term “plant” is used in the context of the present invention,this term also includes any locus conversions of that plant or variety.The term “locus converted plant” as used herein refers to those plantsthat are developed, for example, by backcrossing, genome editing,genetic transformation and/or mutation, wherein essentially all of thedesired morphological and physiological characteristics of a variety arerecovered in addition to the one or more genes introduced into thevariety. To illustrate, backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, e.g.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to the recurrentparent. The parental plant that contributes the gene for the desiredcharacteristic is termed the “nonrecurrent” or “donor parent.” Thisterminology refers to the fact that the nonrecurrent parent is generallyused one time in the breeding e.g., backcross) protocol and thereforedoes not recur. The gene that is transferred can be a native gene, amutated native gene or a transgene introduced by genetic engineeringtechniques into the plant (or ancestor thereof). The parental plant intowhich the gene(s) from the nonrecurrent parent are transferred is knownas the “recurrent” parent as it is used for multiple rounds in thebackcrossing protocol. Poehlman & Sleper (1994) and Fehr (1993). In atypical backcross protocol, the original variety of interest (recurrentparent) is crossed to a second variety (nonrecurrent parent) thatcarries the gene(s) of interest to be transferred. The resulting progenyfrom this cross are then crossed again to the recurrent parent and theprocess is repeated until a plant is obtained wherein essentially all ofthe desired morphological and physiological characteristics of therecurrent parent are recovered in the converted plant in addition to thetransferred gene(s) and associated trait(s) from the nonrecurrentparent.

Genetic Analysis of Watermelon Line 34WA009.

The invention further provides a method of determining a geneticcharacteristic of watermelon line 34WA009 or a progeny thereof, e.g., amethod of determining a genotype of watermelon line 34WA009 or a progenythereof. In embodiments, the method comprises detecting in the genome ofa 34WA009 plant, or a progeny plant thereof, at least a firstpolymorphism (e.g., by detecting a nucleic acid marker by a methodcomprising nucleic acid amplification and/or nucleic acid sequencing).To illustrate, in embodiments, the method comprises obtaining a sampleof nucleic acids from the plant and detecting at least a firstpolymorphism in the nucleic acid sample. Optionally, the method maycomprise detecting a plurality of polymorphisms (e.g., two or more,three or more, four or more, five or more, six or more, eight or more orten or more polymorphisms, etc.) in the genome of the plant. Inrepresentative embodiments, the method further comprises storing theresults of the step of detecting the polymorphism(s) on a computerreadable medium. The invention further provides a computer readablemedium produced by such a method.

DEPOSIT INFORMATION

Applicants have made a deposit of at least 2500 seeds of watermelon line34WA009 with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., 20110-2209 U.S.A. under ATCCDeposit No ______ on ______. This deposit of watermelon variety 34WA009will be maintained in the ATCC depository, which is a public depository,for a period of 30 years, or 5 years after the most recent request, orfor the effective life of the patent, whichever is longer, and will bereplaced if any of the deposited seed becomes nonviable during thatperiod. Additionally, Applicants have satisfied all the requirements of37 C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the samples. Access to this deposit will be made availableduring the pendency of this application to the Commissioner uponrequest. Upon the issuance of a patent on the variety, the variety willbe irrevocably and without restriction released to the public byproviding access to the deposit of at least 2500 seeds of the varietywith the ATCC. Applicants impose no restrictions on the availability ofthe deposited material from the ATCC; however, Applicants have noauthority to waive any restrictions imposed by law on the transfer ofbiological material or its transportation in commerce. Applicants do notwaive any infringement of its rights granted under this patent or underthe Plant Variety Protection Act (7 USC § 2321 et seq.).

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be apparent that certain changes and modifications suchas single locus modifications and mutations, somaclonal variants,variant individuals selected from large populations of the plants of theinstant inbred and the like may be practiced within the scope of theinvention.

1. A seed of tetraploid watermelon line 34WA009, a representative sampleof seed having been deposited under ATCC Accession No. PTA-126985.
 2. Aplant of tetraploid watermelon line 34WA009, a representative sample ofseed having been deposited under ATCC Accession No. PTA-126985.
 3. Awatermelon plant, or a part thereof, having all the physiological andmorphological characteristics of the watermelon plant of claim
 2. 4. Aseed that produces the plant of claim
 3. 5. A plant part of the plant ofclaim 2, wherein the plant part is a fruit, an F1 seed, a scion, arootstock, a shoot, pollen, an ovule, an anther, a root, or a cell.
 6. Atissue culture of regenerable cells of the watermelon plant of claim 2.7. A converted watermelon plant produced by introducing a single locusconversion into the plant of claim 2, wherein said converted watermelonplant comprises said single locus conversion and otherwise comprises allof the physiological and morphological characteristics of watermelonline 34WA009.
 8. A seed that produces the plant of claim
 7. 9. A methodof producing watermelon seed, the method comprising crossing the plantof claim 2 with itself or a second watermelon plant and harvesting theresulting seed.
 10. An F1 seed produced by the method of claim
 9. 11. Awatermelon plant, or a fruit thereof, produced by growing the seed ofclaim
 10. 12. A method of producing a watermelon plant, the methodcomprising growing a diploid reversion of a watermelon plant produced bygrowing the watermelon seed of claim
 1. 13. (canceled)
 14. A method ofdeveloping a watermelon line in a watermelon plant breeding programusing plant breeding techniques, which include employing a watermelonplant, or its parts, as a source of plant breeding material, the methodcomprising: (a) obtaining the watermelon plant, or its parts, of claim 2as a source of breeding material; and (b) applying plant breedingtechniques.
 15. A method for producing a seed of a watermelon plantderived from the plant of claim 2, the method comprising: (a) crossing aplant of watermelon line 34WA009 with a second watermelon plant; (b)allowing seed to form; (c) growing a plant from the seed of step (b) toproduce a plant derived from watermelon line 34WA009; (d) selfing theplant of step (c) or crossing it to a second watermelon plant to formadditional watermelon seed derived from watermelon line 34WA009; and (e)optionally repeating steps (c) and (d) one or more times to generatefurther derived watermelon seed from watermelon line 34WA009, wherein instep (c) a plant is grown from the additional watermelon seed of step(d) in place of growing a plant from the seed of step (b).
 16. A methodof vegetatively propagating the plant of claim 2, the method comprising:(a) collecting tissue capable of being propagated from a plant ofwatermelon line 34WA009; (b) cultivating the tissue to obtainproliferated shoots; and (c) rooting the proliferated shoots to obtainrooted plantlets
 17. The method of claim 16, wherein the method furthercomprises growing plants from the rooted plantlets.
 18. A method ofintroducing a desired added trait into watermelon line 34WA009, themethod comprising: (a) crossing the plant of claim 2 with a watermelonplant that comprises a desired added trait to produce F1 progeny; (b)selecting an F1 progeny that comprises the desired added trait; (c)crossing the selected F1 progeny with watermelon line 34WA009 to producebackcross progeny; (d) selecting a backcross progeny comprising thedesired added trait and otherwise all of the physiological andmorphological characteristics of the watermelon line 34WA009; and (e)optionally repeating steps (c) and (d) one or more times to produce aplant derived from watermelon line 34WA009 comprising a desired addedtrait and otherwise all of the physiological and morphologicalcharacteristics of watermelon line 34WA009, wherein in step (c) theselected backcross progeny produced in step (d) is used in place of theselected F1 progeny of step (b).
 19. A watermelon plant produced by themethod of claim 18 or a selfed progeny thereof, wherein said watermelonplant or selfed progeny thereof has the desired added trait andotherwise has all of the physiological and morphological characteristicsof watermelon line 34WA009.
 20. A seed that produces the plant of claim19.
 21. A method of producing a plant of watermelon line 34WA009comprising a desired added trait, the method comprising introducing atransgene conferring the desired trait into the plant of claim
 2. 22. Awatermelon plant produced by the method of claim 21 or a selfed progenythereof, wherein said watermelon plant or selfed progeny thereofcomprises the transgene and has the desired added trait and otherwisehas all of the physiological and morphological characteristics ofwatermelon line 34WA009.
 23. A seed that produces the plant of claim 22.24. A method of producing triploid watermelon seed, the methodcomprising: (a) crossing the watermelon plant of claim 2 with a diploidwatermelon plant; and (b) harvesting the resultant triploid watermelonseed.
 25. An F1 triploid watermelon seed produced by the method of claim24.
 26. An F1 triploid watermelon plant, or fruit thereof, produced fromthe seed of claim
 25. 27. A method of producing seedless watermelonfruit, the method comprising: (a) crossing the triploid plant of claim26 and a diploid watermelon plant; (b) allowing seedless fruit to form;and (c) harvesting the seedless fruit.
 28. A method of producing atetraploid watermelon plant, the method comprising crossing the plant ofclaim 2 with a different tetraploid watermelon plant.
 29. An F1tetraploid watermelon plant, or a fruit thereof, produced by the methodof claim
 28. 30. A method of determining a genotype of watermelon line34WA009, the method comprising: (a) obtaining a sample of nucleic acidsfrom the plant of claim 2; and (b) detecting a polymorphism in thenucleic acid sample.